Age related macular degeneration (AMD) is the leading cause of irreversible vision loss in the United States and many other industrialised countries. “Dry” AMD is the most common type of macular degeneration and affects 90% of the people who have the condition. The dry form is characterized by the formation of drusen within the macula, a specialized structural region of the retina which capture the light that enters the eye. Typically, drusen is formed under the retinal pigment epithelial (RPE) cells and its presence is thought to lead to atrophy of photoreceptors due to a breakdown or thinning of the RPE layer of that supports the photoreceptor cells. It is also thought that persistence of drusen within the retina leads to a persistent inflammatory reaction and results in a cascade of secondary responses that eventually can lead to wet AMD.
The “wet” form of AMD is characterized by an abnormal outgrowth of blood vessels from the vasculature situated behind the retina in a process that is often referred to as choroidal neovascularization (CNV). While not as prevalent as the dry form, it has a more rapid onset and is more severe phenotype, often leading to reduction of a substantial portion of the visual field.
The current standard of care for wet AMD is Ranibizumab (RAN), a monoclonal antibody fragment with strong affinity to the vascular endothelial growth factor-A (VEGF-A), a molecular moiety secreted from cells and known to cause the formation or growth of nascent blood vessels. RAN binds to and inhibits the biologic activity of VEGF-A, thereby preventing the interaction of VEGF-A with its receptors (VEGFR1 and VEGFR2) on the surface of endothelial cells. This results in a reduction in endothelial cell proliferation, less vascular leakage, and a reduction in new blood vessel formation characteristic of CNV.
The ocular half-life of RAN, however, is only nine days following intravitreal injection, thus therapeutic doses must be administered monthly to patients to remain effective at suppressing vascular proliferation. Although useful at stabilizing visual acuity in nearly 95% of patients, improved vision was noted in only 29%-40% of patients. RAN acts as a molecular sponge to mop-up secreted VEGF-A. Inefficiencies in this process may be one reason why vision is only stabilized, not improved in most patients. In other words, it treats the symptoms but not the cause.
The principal drawback with existing monoclonal antibody wet AMD therapies is the requirement for frequent, continuous treatment, typically involving monthly injections into the eye. Combined with a rapidly aging population and correspondingly low numbers of clinicians who are qualified to administer intravitreal injections, application of this therapy This has placed enormous strain on healthcare systems. Thus there is clearly a need for longer lasting treatments and/or treatments that can reverse the symptoms. Alternative treatments for wet AMD have been similarly unsatisfactory, also as a result of their frequency of administration, but as well as their side effects or poor efficacy.
One of the newer drugs to commence clinical trials is that of the VEGF Trap Eye (VTE) which incorporates the second binding domain of the VEGFR-1 receptor and the third domain of the VEGFR2 receptor 1. By fusing these extracellular protein sequences to the Fc segment of a human IgG backbone, developers have created a chimeric protein with a very high VEGF binding affinity (Stewart M W. Br J Ophthalmol (2012). doi:10.1136/bjophthalmol-2011-300654). As well as binding all isomers of the VEGF-A family, it also binds VEGF-B and placental growth factor.
Given the fact that the chimera protein still has a relatively short half-life, VTE however must still be regularly administered—every 2 months.
AAV2-sFLT01 is a gene therapy vector that expresses a modified soluble Flt1 receptor coupled to a human IgG1 Fc. As a high affinity VEGF binding protein, AAV2-sFLT01 functions to neutralize the pro-angiogenic activities of VEGF for treatment of wet AMD via an intravitreal injection. (Wasworth et al. Molecular Therapy vol. 19 no. 2 Feb. 2011; 326-334). The use of an AAV vector is expected to ensure long-term expression, lasting for many months or even years, from a single injection. However, in order to accommodate the sFLT01 and IgG1 Heavy Chain Fc fusion protein, single stranded AAV must be used, which in turn requires high quantities of vector for efficient transduction and thus increases the risk of an immune response to the viral capsid proteins. Moreover, a high prevalence of the normal adult population has been exposed to serotype 2 variant of AAV, and may have pre-existing immunity against it.
The molecule PF-04523655 is a 19 nucleotide siRNA that inhibits the expression of the hypoxia-inducible gene RTP801 (Nguyen et al. Ophthalmology. 2012 September; 119(9):1867-73). In clinical studies conducted to date, it has been found to prevent neovascularization and vessel leakage, although does so via a different pathway than VEGF. It has been demonstrated that the siRNA only persists in the eye for several weeks, meaning that like so many of the other existing and developing therapies, patients will require regular intravitreal injections for treatment. A failure to do so with many treatments has seen a continued loss of visual acuity, and a progression of degeneration.
More generally, previous siRNA-based approaches for treating and managing wet AMD have failed. Although initial pre-clinical experimental results were encouraging, it was subsequently demonstrated that mode of action of these molecules was not through a sequence specific RNAi-based mechanism, but rather through induction of a non-specific interferon response mediated by the interaction of siRNAs with Toll-like receptor TLR3 (Kleinmann et al 2008). Toll-like receptors are transmembrane proteins that play a key role in the innate immune system. Often positioned on either the cell surface or on intracellular vesicles such as the endosome, some family members of this family recognize double stranded RNA, not normally present in the endogenous cell, as foreign substance and triggers a cascade of molecule responses. This leads to interferon activation, which has a transitory therapeutic effect in mouse models. However interferon has a much lower efficacy in humans which explains the poor efficacy of this treatment in human clinical testing.
Retinostat is an equine infectious anaemia virus (EIAV) based lentivirus vector expressing angiostatin and endostatin, both of which are naturally occurring angiogenesis inhibitors in the ocular compartment. Endostatin blocks VEGF signalling, reduces vascular permeability, decreases cell matrix adhesion and promotes endothelial cell apoptosis. Angiostatin prevents endothelial cell proliferation and migration. The genes are delivered via a subretinal injection and inhibit the formation of new blood vessels. Sub-retinal delivery however requires an intensive surgical procedure, which, unlike intravitreal delivery, does not lend itself to outpatient treatments or treatment at a local doctor.
Despite the large amount of development activity in the field of AMD therapeutics, and wet AMD in particular, there remains a need to create more effective therapies that are also patient friendly with respect to side effects, the mode of treatment and the frequency thereof. This invention is directed to a RNA interference (RNAi) agent and the use of that RNAi agent to manage and treat wet AMD in individuals.
The RNAi pathway is initiated by the enzyme Dicer, which cleaves double-stranded RNA (dsRNA) molecules into short fragments (commonly referred to as siRNAs) of ˜20-25 nucleotides. One of the two strands of each fragment, known as the guide strand or active strand, is then incorporated into the RNA-induced silencing complex (RISC) through binding to a member of the Argonaute protein family. After integration into the RISC, the guide strand base-pairs with its target mRNA and is thought to either inhibit a target by inhibiting translation (by stalling the translational machinery) and/or inducing cleavage of the mRNA, thereby preventing it from being used as a translation template.
While the fragments produced by Dicer are double-stranded, only the guide strand, directs gene silencing. The anti-guide strand (referred to commonly as a passenger strand, carrier strand or * strand) is frequently degraded during RISC activation (Gregory R et al., 2005). RISC assembly is thought to be governed by an enzyme that selects which strand of a dsRNA Dicer product is loaded into RISC. This strand is usually the one whose 5′ end is less tightly paired to its complement. There also appears to be a clear bias for A, and to a lesser extent U, at the 5′ position to facilitate binding to some Argonaute proteins (Schwarz D S et al., 2003; Frank F et al., 2010).
The present invention seeks to overcome the problems associated with other therapies as already discussed above, while overcoming the previous challenges faced by RNAi therapeutics in this field.
Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.